: In the previous grant period, we developed a multicomponent DNA carrier that, through the incorporation of endosomolytic peptides, is capable of releasing targeted nucleic acids directly to the cytoplasm of hepatocytes. Using multicomponent carriers, the efficiency of delivery of nucleic acids into cells was increased by 3 orders of magnitude, and the amount of DNA introduced into the nuclei in animals was increased 1 to 2 orders of magnitude compared to the originally described system. Recently, we have developed new approaches to enhance integration of targeted genes in cell culture resulting in persistent transgene expression. The objective of the current project is to take advantage of these advances to evaluate a novel therapeutic strategy against hepatitis B viral infection: transfection-mediated therapeutic selection. The hypothesis is: integrative transfection of antiviral genes will result in continuous inhibition of HBV gene expression, and block production of viral antigens. Under the pressure of host cell-mediated immune response against cells bearing HBV antigens, there will be a selective survival advantage to transfected cells and their progeny. The antiviral agents are proposed to be generated from genes whose products are directed against unique features of the HBV life cycle, blocking translation, and replication. These areas distinguish viral processes from those of the mammalian host in order to achieve efficacy without toxicity. Candidates will be screened using stable HBV expression models in cell culture first to identify optimal constructs. Agents will be targeted to liver by complexation with multicomponent carriers, and also by genetic engineering into chimeric adeno-associated viral vectors all with integration enhancement capability. After optimization and comparison in an HBV cell culture model system, antiviral agents will be used to test the therapeutic approach in an animal model of HBV infection. Agents will be prepared against analogous sequences in the Woodchuck Hepatitis Virus (WHV), a homologous hepadna virus. Efficacy of long-term inhibition of HBV gene expression and replication, and evidence of selective survival advantage of cells bearing integrated antiviral genes will be determined over the time course, and optimal agents compared.